Adenovirus-mediated Transfer of Fas Ligand Gene Augments Radiation-induced Apoptosis in U-373MG Glioma Cells

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.     

Co-transduction of Apaf-1 and caspase-9 augments etoposide-induced apoptosis in U-373MG glioma cells.

Several apoptosis-related genes have been reported to be involved in chemotherapy-induced apoptosis in cancers. An assessment of the relationship between expression of those genes and the degree of chemotherapy-induced apoptosis may be useful in improving the efficacy of cancer therapy. We transduced Apaf-1 (apoptotic protease-activating factor-1) and caspase-9 into U-373MG glioma cells using adenovirus (Adv) vectors in the presence of etoposide and evaluated the degree of apoptosis. The degree of apoptosis in etoposide-treated U-373MG cells infected with Adv for Apaf-1 (Adv-APAF1) was higher (27%) than that in cells infected with control Adv (14%), that in cells infected with Adv for caspase-9 (Adv-Casp9) was higher (34%) than that in cells infected with Adv-APAF1, and that in cells infected with both Adv-APAF1 and Adv-Casp9 was the highest (41%). Treatment with etoposide increased expression of p53 and decreased expression of Bcl-X(L) in U-373MG cells which harbored mutant p53. These results indicate that the expression of Apaf-1 and caspase-9 may be important determinants in predicting the sensitivity of cancers to chemotherapy. Adv-mediated co-transduction of Apaf-1 and caspase-9 should render cancer cells highly sensitive to chemotherapy.     

Co-transduction of Apaf-1 and caspase-9 highly enhances p53-mediated apoptosis in gliomas

Mutation of the p53 gene plays a critical role in the development of cancer and response to cancer therapy. To analyze the mechanism of cancer development and to improve cancer therapy, it is important to assess which genes are downstream components of p53 in cancers, and whether the expression levels of these genes affect p53-mediated apoptosis. In this study, we transduced the wild type p53 gene along with the Apaf-1 and caspase-9 genes via adenovirus vectors into U251 and U-373MG glioma cells harbouring a mutated p53, and evaluated the degree of apoptosis. Co-induction of Apaf-1 and caspase-9 genes highly enhanced p53-mediated apoptosis in glioma cells. Induction of wild type p53 enhanced the expression levels of Bax, p21/WAF1, and Fas protein. To determine which gene is activated by wild type p53 induction and, in turn, activates Apaf-1 and caspase-9, we transduced the Bax, p21/WAF1 or Fas gene via adenovirus vector to U251 cells to achieve a similar expression level as that induced by the Adv for p53 in U251 cells. U251 cells transduced with Fas concomitant with the Apaf-1 and caspase-9 genes underwent drastic apoptosis. This suggests that induction of wild type p53 upregulates Fas, which in turn may play a role in the activation of Apaf-1 and caspase-9. These results are important for analyzing the mechanism of tumour development and for predicting the therapeutic effect of p53 replacement gene therapy in a particular patient.     

莱菔硫烷促进神经胶质瘤细胞凋亡的机制探讨

目的探讨莱菔硫烷(SFN)促进神经胶质瘤细胞凋亡的作用机制。方法采用噻唑蓝(MTT)法与流式细胞法检测不同浓度的SFN对神经胶质瘤U251细胞系生长的抑制作用,测定半数抑制浓度,空白对照设为对照组;采用Western blot研究SFN对U251细胞内Cyt-c的表达情况。结果不同浓度SFN组A值均低于对照组,差异有统计学意义(P<0.05)。不同浓度SFN均对U251细胞生长有一定抑制作用,12.5μmol/l、25μmol/l、50μmol/l、100μmol/l浓度的SFN对U251细胞生长抑制率逐渐增强,各组间比较差异均有统计学意义(P<0.05)。但100μmol/l与200μmol/l浓度的SFN对U251细胞生长抑制率比较无统计学意义(P>0.05)。不同浓度SFN诱导细胞凋亡率及Cyt-c蛋白表达均显著高于对照组,且随着SFN浓度递增,U251细胞凋亡率、Cyt-c蛋白表达逐渐增加,各组间比较差异均有统计学意义(P<0.05)。结论SFN可能参与神经胶质瘤细胞的凋亡过程,表现为诱导凋亡U251细胞,可能与Cyt-c表达增高存在关系。     

Tumor Necrosis Factor-α Gene Transfer Augments Anti-Fas Antibody-mediated Apoptosis in Human Glioma Cells

To effectively induce apoptosis in human glioma cells, we tried to transfer the tumor necrosis factor (TNF)-α gene into glioma cells to produce TNF-α locally in these cells. The stable transfectants of three gliorna cells (U251-SP, U251-MG, and T98G) were resistant to exogenous TNF-α, but their cell surface expression of the Fas antigen was dramatically enhanced by about 10 to 100-fold as compared with untransfected glioma cells exposed to exogenous TNF-α. The Fas antigen is a transmemhrane cytokine receptor protein of the nerve growth factor/TNF receptor superfamily. Although the untransfected glioma cells tested were resistant to anti-Fas antibody-mediated apoptosis, the TNF-α gene-transfected glioma cells exhibited high susceptibility to anti-Fas antibody-mediated apoptosis. Thus, TNF-α gene transfer combined with anti-Fas antibodies may be useful for the treatment of malignant glioma.     

Over-expression of APAF-1 and caspase-9 augments radiation-induced apoptosis in U-373MG glioma cells.

The p53 tumor-suppressor gene plays a critical role in radiation-induced apoptosis. Several genes, including Bax and Fas, are involved in p53-mediated apoptosis, and their over-expression enhances the degree of radiation-induced apoptosis. Apaf-1 and caspase-9 have been reported to be downstream components of p53-mediated apoptosis, suggesting that these genes play a role in radiation-induced apoptosis. In this study, we transduced U-373MG cells harboring mutant p53 with the Apaf-1 and/or caspase-9 genes via adenoviral (Adv) vectors concomitant with X-ray irradiation and evaluated the degree of apoptosis. The percentage of apoptotic cells in U-373MG cells co-infected with the Adv for Apaf-1 (Adv-APAF-1) and that for caspase-9 (Adv-Casp9) and treated with irradiation (24%) was much higher than that in cells co-infected with Adv-APAF-1 and Adv-Casp9 and not treated with irradiation (0.86%) and that in cells infected with either Adv-APAF-1 or Adv-Casp9 and treated with irradiation (2.0% or 2.6%, respectively). The apoptosis induced by co-transduction of Apaf-1 and caspase-9 and irradiation was repressed in cells that were co-infected with the Adv for Bcl-X(L) but not in cells co-infected with the Adv for Bcl-2. These results indicate that Apaf-1 and caspase-9 play a role in radiation-induced apoptosis in cancer cells harboring mutant p53. Bcl-X(L) may be critically involved in the radioresistance of cancer cells by repressing Apaf-1- and caspase-9-mediated apoptosis. Expression of Apaf-1 and caspase-9 in tumors may be an important determinant of the therapeutic effect of irradiation in cancer treatment.     

慢病毒载体介导RNAi稳定抑制VASH1表达对人脑胶质瘤细胞增殖、凋亡影响的体外研究

目的 探讨运用慢病毒载体介导的RNA干扰技术抑制血管生成抑制因子1（VASH1）的表达对人脑胶质瘤U-87MG细胞增殖和凋亡的影响。方法 应用pGCL-GFP质粒构建针对VASH1的慢病毒shRNA载体,转染入工具细胞293T,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞;通过RT-PCR和Western blot评价其对U-87MG细胞内VASH1表达的影响;用四甲基偶氮唑蓝（MTT）法观察U-87MG细胞增殖活性的改变;用流式细胞仪（FCM）检测U-87MG细胞凋亡的变化。结果 与转染阴性对照组和空白组相比,转染pGCL-GFP-VASH1慢病毒载体的U-87MG细胞VASH1的mRNA 蛋白表达水平明显降低（P<0.01）;细胞的增殖活性显著上调（P<0.01）,细胞凋亡明显减少（P<0.01）。结论 VASH1 shRNA慢病毒载体可抑制VASH1在人脑胶质瘤U-87MG细胞中的表达,并增加肿瘤细胞的增殖活性,抑制细胞凋亡。     

白藜芦醇抑制人脑胶质瘤细胞生长及诱导凋亡比较

 目的 探讨白藜芦醇（Resveratrol，Res）体外抑制U251、U87和SHG-44脑胶质瘤细胞生长及诱导凋亡的不同作用比较，验证Re8确可导致U251细胞凋亡。方法 MTT比色法测量不同剂量的Res作用U251、U87和SHG-44胶质瘤细胞24h后对增殖的影响及不同剂量的Res作用U251、U87和SHG-44细胞6h、24h、48h后的影响。流式细胞仪用Annexin V-FITC和PI双染检测U251、U87和SHG-44细胞凋亡率。HE染色、Hoechst 33342荧光染色、透射电镜（TEM）观察U251细胞形态改变，流式细胞仪测定U251细胞的细胞周期改变。结果 Res明显抑制U251、U87和SHG-44细胞的生长和增殖（P〈0．01）且对U87细胞的抑制作用最强，U251和SHG-44细胞次之；对U251细胞的抑制作用呈浓度及时间依赖性反应；Re8所致的U251、U87和SHG-44胶质瘤细胞凋亡为浓度依赖关系，且对U87细胞的凋亡作用强于U251和SHG-44细胞。U251凋亡细胞的细胞周期主要发生G，期阻滞。结论 Res对U251、U87和SHG-44细胞生长抑制及凋亡作用以U87细胞更明显，对U251细胞的抑制作用呈浓度及时间依赖性反应并引起了其细胞周期改变。     

miR-374a Inhibitor Enhances Etoposide-Induced Cytotoxicity Against Glioma Cells Through Upregulation of FOXO1

Glioma is a commonly diagnosed brain tumor that shows high mortality rate. Despite the great advancement of cancer therapy in recent years, chemotherapy is still an important approach for treatment of glioma. However, long-term chemotherapy usually causes serious side effects or complications. It is desirable to take strategies to enhance the efficacy of current chemotherapy. In the present study, we observed obvious upregulation of miR-374a in glioma cells. More importantly, we found that knockdown of miR-374a was able to enhance the etoposide-induced cytotoxicity against glioma cells. Mechanically, we demonstrated that FOXO1 was the target of miR-374a in glioma. Treatment with miR-374a inhibitor induced overexpression of FOXO1, and thus promoted the expression of Bim and Noxa. Since Bim and Noxa act as key proapoptotic proteins in mitochondrial apoptosis, miR-374a inhibitor was able to enhance the etoposide-induced apoptosis pathway in glioma.     

人参皂甙Rh2诱导C6胶质瘤细胞凋亡的实验研究

目的:观察不同浓度人参皂甙Rh2(G-Rh2)对C6胶质瘤细胞的诱导凋亡作用,并在分子水平探讨其作用机理。方法:细胞计数法检测细胞增殖,流式细胞仪分析细胞的DNA组织含量变化,电镜下观察细胞的超微结构改变。结果:1)不同浓度G-Rh22、4、8μmol/L对C6胶质瘤细胞有增殖抑制作用,且呈明显量效关系;2)流式细胞术分析经G-Rh2作用72h的C6胶质瘤细胞的DNA组织直方图提示有凋亡发生;3)不同浓度G-Rh22、4、8μmol/L可使G0/G1期细胞比率下降(P<0.05,P<0.01),S期细胞比率明显升高(P<0.05,P<0.01),而G2/M期细胞相对稳定(P>0.05);4)电镜结果提示经4μmol/LG-Rh2作用72h的C6细胞呈典型的凋亡形态学改变。结论:G-Rh2对C6胶质瘤细胞有诱导凋亡作用,呈剂量依赖性抑制C6胶质瘤细胞增殖,对脑胶质瘤的治疗有重要意义。     

卵巢浆液性肿瘤中凋亡调控因子Smac Caspase-9和Caspase-3的表达及意义

目的:探讨凋亡调控因子Smac、Caspase-9和Caspase-3在卵巢浆液性肿瘤中的表达。方法:采用免疫组织化学技术(SP法)和图像分析技术检测25例卵巢浆液性囊腺癌、10例卵巢交界性浆液性囊腺瘤、30例卵巢浆液性囊腺瘤和24例正常卵巢组织中凋亡调控因子Smac、Caspase-9和Caspase-3表达水平。结果:Caspase-3、Caspase-9和Smac在30例浆液性囊腺瘤中的表达率分别为93%、93%和73.3%;在10例交界性浆液性囊腺瘤中的表达率分别为70%、80%和80%;在25例浆液性囊腺癌中的表达率分别为96%、96%和84%。阳性信号主要定位于细胞质,呈弥漫分布。而在正常卵巢24例中的表达率分别为83.3%、80.9%和58.3%。凋亡调控因子Caspase-3、Caspase-9和Smac在浆液性卵巢肿瘤中的表达增加(P<0.05)。结论:凋亡调控因子Caspase-3、Caspase-9和Smac在卵巢浆液性肿瘤中的表达增加。这提示Caspase-3、Caspase-9和Smac可能会成为卵巢浆液性肿瘤治疗的新靶点。     

Co-transduction of p27Kip1 strongly augments Fas ligand- and caspase-8-mediated apoptosis in U-373MG glioma cells

BACKGROUND: p27Kip1 is a potential tumor suppressor gene. As malignant gliomas express Fas at high levels, the relationship between Fas-mediated apoptosis and p27Kip1 expression may improve therapeutic approaches for treating gliomas. MATERIALS AND METHODS: In this study, we transduced U-373MG glioma cells with the Fas ligand or caspase-8 genes using adenovirus vectors after transduction of the p27Kip1 gene to induce cell cycle arrest in U-373MG cells, and evaluated the degree of apoptosis. RESULTS: The results demonstrate that expression of p27Kip1 enhanced Fas ligand- or caspase-8-mediated apoptosis in U-373MG cells. Expression of apoptosis-related genes such as Bax, Bcl-X(L), Bcl-2 or caspase-8 were reduced by p27Kip1 transduction compared with that of beta-actin, whereas p27Kip1 transduction did not affect the expression level of Fas or the Fas ligand. CONCLUSION: Combined transduction of p27Kip1 with Fas ligand or caspase-8 would overide the resistance mechanism to apoptosis in malignant gliomas.     

整合素与胶质瘤细胞凋亡

 目的　研究整合素 β1在胶质瘤细胞中的表达及在胶质瘤细胞增殖、生存方面的作用。 方法　流式细胞仪检测SHG4 4胶质瘤细胞整合素 β1的含量 ,以不同浓度的抗整合素 β1抗体处理在胶原蛋白I上培养的SHG4 4胶质瘤细胞 ,MTT法测定ID50 ,测定细胞生长曲线 ,流式细胞仪分析细胞周期改变。结果　流式细胞仪检测SHG4 4胶质瘤细胞表达整合素 β1,MTT曲线测定发现 ,2 0 μg/ml抗整合素 β1抗体处理SHG4 4胶质瘤细胞 4 8小时后 ,显著抑制SHG4 4胶质瘤细胞的增殖。流式细胞仪分析可检出凋亡峰。结论　整合素 β1在SHG4 4胶质瘤细胞中高表达 ,并与胶质瘤细胞增殖、生存密切相关 ,抑制整合素 β1的表达可促进胶质瘤细胞的凋亡及坏死。     

The Circadian Gene Per1 Plays an Important Role in Radiation-Induced Apoptosis and DNA Damage in Glioma

Objective: Period1 (PER1), a core circadian gene, not only modulates circadian rhythm but may also play animportant role in other biological processes, including pathways involved in the proliferation and apoptosis of tumorcells. In this study, we investigated the mechanism by which the downregulated expression of PER1 promotes theapoptosis of wild-type P53 human glioma U343 cells exposed to X-rays. Methods:U343 cells were exposed to 6 mV10 Gy X-ray irradiation after infection with an shRNA lentivirus to reduce the expression of PER1 and were analyzedby SCGE analysis, flow cytometry, qRT-PCR, and western blotting. Result: SCGE analysis revealed that compared withthe controls, U343 cells expressing low levels of PER1 showed minor DNA damage when exposed to X-ray irradiation(P<0.05), and the flow cytometry assay showed lower death rates (P<0.05). RT-PCR and western blot analysis bothrevealed decreased expression of CHK2 and P53, which regulate DNA damage and repair via the CHK2-P53 pathway,and decreased expression of C-MYC, which is related to cell apoptosis. Conclusion:Our research suggests that PER1may play an important role in tumor radiotherapy, which is attributable to enhanced chk2-P53 signaling and proapoptoticprocesses. These findings provide a new target for the clinical treatment of glioma and a reliable basis for postradiationtherapy and gene therapy for glioma and other cancers.     

选择性COX-2抑制剂Celecoxib诱导胶质瘤细胞凋亡作用机制初探

背景与目的：选择性COX-2抑制剂具有诱导多种肿瘤细胞凋亡的作用，但在胶质瘤方面研究还不多见，本研究门的是探讨选择忡COX-2抑制剂塞来昔布（Celecoxib）诱导胶质瘤C6细胞凋亡，及其在诱导胶质瘤细胞凋亡的作用机制方法：应州PGE2检测、吖啶橙染色、流式细胞仪（FCM）检测不同浓度的Celecoxib对胶质瘤C6细胞作用，通过放免法检测不同浓度Celecoxib作用于胶质瘤C6细胞后PGE2的含量，通过吖啶橙染色从形态上观察治疗组细胞的形态学变化，流式细胞仪检测各组细胞发生凋亡的情况。结果：随Celecoxib浓度增加而PGE2合成减少（P〉0．05）．吖啶橙染色荧光染色对照组细胞核DNA为均匀黄色或黄绿色的荧光，治疗组（Celecoxib 50μmol／L）凋亡细胞的细胞核或细胞质内可处致密浓染的黄绿色荧光或黄绿色碎片．Celecoxib浓度为12．5μmol／L和50μmol／L时．FCM法检测凋亡分别为5．83％和15．32％．Celecoxib对C6胶质瘤细胞增殖抑制作用具有浓度依赖性．细胞凋亡随浓度增加而增多。结论：Celecoxib具有诱导胶质瘤C6细胞凋亡的作用并呈剂量依赖性，随PGE2合成下降细胞凋亡增多，PGE2途径是Celecoxib诱导胶质瘤细胞凋亡机制之一．对胶质瘤的治疗有重要意义。     

Effects of Celecoxib on Cycle Kinetics of Gastric Cancer Cells and Protein Expression of Cytochrome C and Caspase-9

Objective: This investigation aimed to determine effects of celecoxib on the cell cycle kinetics of the gastriccancer cell line MGC803 and the mechanisms involved by assessing expression of cytochrome C and caspase-9at the protein level. Methods: Cell proliferation of MGC803 was determined by MTT assay after treatment withcelecoxib. Apoptosis was assessed using fluorescence staining and cell cycle kinetics by flow cytometry. Westernblotting was used to detect the expression of caspase-9 protein and of cytochrome C protein in cell cytosol andmitochondria. Results: Celecoxib was able to restrain proliferation and induce apoptosis in a dose- and timedependentmanner, inducing G0/G1 cell cycle arrest, release of cytochrome C into the cytosol, and cleavageof pro-caspase-9 into its active form. Conclusion: Celecoxib can induce apoptosis in MGC803 cells through amechanism involving cell cycle arrest, mitochondrial cytochrome C release and caspase activation.     

Phosphorylation of Fas-associated Death Domain Contributes to Enhancement of Etoposide-induced Apoptosis in Prostate Cancer Cells

Fas-associated death domain (FADD) plays an important role as an adapter molecule in Fas (CD95/APO-l)-mediated apoptosis and contributes to anticancer drug-induced cytotoxicity. We treated three human prostate cancer cell lines with etoposide, a toposiomerase II inhibitor with activity against various tumors including prostate cancer. We found that the overexpression of FADD sensitizes etoposide-induced apoptosis through a rapid activation of c-Jun NH₂-terminal kinase (JNK) and, subsequently, of caspase 3. In addition, phosphorylation of FADD at serine 194 coincided with this sensitization. Treatment with the caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), or overexpression of either mitogen-activated protein kinase kinase (MKK) 7 or Bcl-xL canceled FADD-mediated sensitization to etoposide-induced apoptosis. Moreover, treatment with the caspase 8 inhibitor, benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (z-IETD-fmk), or overexpression of viral FLICE/caspase-8-inhibitory protein (FLIP) from equine herpesvirus type 2 E8 also had an inhibitory effect, supporting a major involvement of a caspase 8-dependent mitochondrial pathway. Interestingly, FADD was phosphorylated, and etoposide-induced JNK/caspase activation and apoptosis were enhanced in the cells arrested at G2/M transition, but not in those overexpressing mutant FADD, in which 194 serine was replaced by alanine. Our results demonstrate that phosphorylated FADD-dependent activation of the JNK/caspase pathway plays a pivotal role in sensitization to etoposide-induced apoptosis in prostate cancer cells.     

全反式维甲酸诱导大鼠C6脑胶质瘤细胞凋亡的实验研究

目的: 探讨全反式维甲酸对大鼠C6脑胶质瘤细胞的增殖抑制及其分子机制。 方法: MTT法检测全反式维甲酸作用于大鼠C6脑胶质瘤细胞后,观察其对细胞增殖抑制率的影响。流式细胞仪观察肿瘤细胞周期及凋亡率的变化。电镜观察C6细胞超微结构变化。Western blot法在不同时间点对凋亡相关基因caspase-3活性蛋白产物的表达进行了检测。 结果: MTT结果表明ATRA对C6细胞的抑制作用具有时间和浓度依赖性。流式细胞仪检测证明与对照组相比,处理组C6细胞发生G₁期阻滞;S、G₂期细胞比例下降;细胞出现亚二倍峰,凋亡比例明显增加。电镜下全反式维甲酸作用72h后处理组C6细胞呈凋亡改变:如核固缩、染色质趋边凝聚。Western blot检测发现,处理组出现了caspase-3蛋白活性裂解片段。 结论: 全反式维甲酸抑制C6脑胶质瘤细胞生长,全反式维甲酸抑制脑胶质瘤的作用机理可能至少通过改变细胞周期分布、诱导凋亡来实现。     

去甲二氢愈创木酸诱导体外恶性胶质瘤细胞凋亡的研究

目的：观察去甲二氢愈创木酸（nordihydroguaiaretic acid,NDGA)对人恶性胶质瘤细胞系SHG-44细胞凋亡的影响,并探讨其发生的可能机理。方法：采用MTT（methy thiazolyl tetrazolium)法检测细胞增殖抑制的情况。利用光镜和电镜观察及TUNEL法检测体外培养细胞的凋亡情况,用免疫组织化学,原位杂交和图像分析等方法检测bcl-2基因mRNA和蛋白的表达水平。结果：（1）一定浓度（100μmol/L）的NDGA处理SHG-44细胞不同时间后,在NDGA抑制细胞增殖的同时,也可诱导此细胞发生凋亡,且随着作用时间的延长,凋亡细胞数增加越明显;（2）免疫组化染色结果表明,一定浓度的NDGA处理细胞不同时间后,出现SHG-44细胞bcl-2蛋白表达水平降低,且随着作用时间的延长,这种趋势更加明显,与细胞凋亡的发生呈负相关;（3）原位杂交结果显示;NDGA处理细胞不同时间后,出现SHG-44细胞bcl-2基因mRNA的表达水平降低,结果与免疫组化检测一致。结论：NDGA可诱导SHG-44细胞的凋亡,这种作用可能与其下调bcl-2基因的表达有关,确切机制有待进一步深入研究。